Study species and seed material
We focused on Biscutella didyma L., a winter annual Brassicaceae
with Mediterranean and Irano-Turanian distribution (Feinbrun-Dothan &
Danin 1998). This medium-sized, common and subordinate species was
selected because it is one of few species
occurring reliably across the
entire rainfall gradient. As is typical for many annuals in our study
region, B. didyma has short dispersal distances (Siewert &
Tielbörger 2010) and low outcrossing rates
(Boaz et al. 1990), making
gene-flow among manipulated plots unlikely.
First, seeds of 100 B. didyma individuals (genotypes) per
rainfall treatment and site were collected in spring 2012 by randomly
picking twenty individuals within each 10m × 25m plot. Keeping ≥1.5m
distance among genotypes and the large plot size ensured independence
and capturing a representative fraction of the intrapopulation variance.
One offspring per genotype was raised with regular watering for one
generation in Tübingen (Germany)
to reduce potential parental
effects and amplify seed material. The growth period (Nov 2012 – May
2013) approximated the natural vegetation season and photoperiod, and
greenhouse temperatures mirrored day temperatures in the field sites
(15–20°C in winter, gradually warming to 25–30°C in spring). Wrapping
flowering plants in light, transparent fabric (organza) ensured
self-pollination and facilitated seed collection separately per mother.
Second, of the resulting second generation,
we randomly picked 40 genotypes
per rainfall treatment and site (3×40 in SA, M; 40 in A, MM; altogether
320 genotypes) for the subsequent
greenhouse experiment (detailed scheme: Appendix Fig. S1).